Can I use Taq polymerase to generate my gene of interest for directional TOPO cloning?
No, your gene of interest must be amplified with a proofreading polymerase such as Platinum SuperFi DNA Polymerase or AccuPrime Pfx DNA Polymerase that leaves blunt ends for directional TOPO cloning.
Which nucleotide analogues can be used with Platinum SuperFi DNA Polymerase?
Platinum SuperFi DNA Polymerase cannot read dUTP-derivatives or dITP in DNA templates, so the use of these analogues is not recommended.Platinum SuperFi DNA Polymerase can incorporate 7-deaza-dGTP and radiolabeled dNTPs.
I am having difficulties amplifying long targets using Platinum SuperFi DNA Polymerase. What are your recommendations?
Good lab practices are important for long fragment amplification. These include using high-quality templates (pure, fresh and intact) and fresh primer solutions. Optimization steps to consider include decreasing denaturation and extension temperatures, lengthening extension times as recommended in the manual, and increasing template amounts.
Can Platinum SuperFi DNA Polymerase amplify AT-rich targets?
Yes. To improve amplification of AT-rich targets, we recommend reducing the extension temperature to 68 degrees C or add 5-15 mM Tetramethylammonium Chloride (TMAC).